Estrogen, acting through its nuclear receptor, stimulates breast development and can enhance the growth of breast cancer. Drugs that interfere with estrogen receptor (ER) action are an important part of breast cancer treatment and are also being used for breast cancer prevention. There has been rapid progress in our understanding of how the ER acts to regulate target genes. Structural studies have provided important insights into how ligands, such as natural estrogens or the synthetic agonist diethylstilbestrol, function as agonists whereas others, such as raloxifene, tamoxifen or ICI compounds, function as antagonists in some tissues. We hypothesize that the ER modulates gene transcription by two fundamentally different mechanisms: (1) a classical estrogen response element (ERE)-mediated pathway and (2) a non-classical pathway that involves ER interactions with other transcription factors present on target genes. Agonists such as estrogen stimulate gene expression by the classical pathway, but repress gene expression through the non-classical pathway. Unraveling the molecular basis of these ER- dependent pathways is fundamental to understanding the role of ER in breast cancer and the pharmacological actions of drugs used to treat or prevent breast cancer. In the current proposal we plan to (1) develop in vitro models of classical and non-classical ER-dependent transcription, (2) develop an in vivo knock-in mutation of ER alpha that discriminates its function through the classical and non-classical pathways, and (3) identify breast epithelial cell target genes in the non-classical ER pathway.